The quantitative determination of mono-, di-, and particularly triglycerides in the body fluids of animals and man has been used in the clinical diagnosis of many diseases or disorders such as artherosclerosis, diabetes mellitus, nephrosis, biliary obstruction, and various metabolic derangements due to endocrine disturbances. Clinical analysis generally requires that the glycerol esters first be hydrolyzed to liberate glycerol and the corresponding fatty acids. Various techniques known in the art such as spectrophotometry are then used to determine the amount of glycerol or fatty acids released by the hydrolysis.
Until recently all of the methods used for the hydrolysis of triglycerides in a body fluid such as serum split the glycerol esters with a strong alkaline solution such as sodium or potassium hydroxide or, alternatively, employed a transesterification agent. These methods required the tedious and time-consuming separation of the lipids from the other serum constituents prior to hydrolysis and were not satisfactory for rapid routine clinical analysis. Recently, a number of enzymatic techniques have been developed for the hydrolysis of serum triglycerides. However, despite the convenience of enzymatic techniques compared to the older methods a number of problems remain.
The first lipase tried in the hydrolysis of serum triglycerides was pancreatic lipase which is isolated from the mammalian pancreas. This enzyme was found to be unsatisfactory because it was unable to completely hydrolyze the serum triglycerides in a reasonable period of time. A lipase isolated from the mold Rhizopus arrhizus (R. arrhizus) has been shown to completely hydrolyze the serum triglycerides within a relatively short period of time. U.S. Pat. No. 3,759,793. The free glycerol was then determined by a series of enzymatic reactions. This technique suffered from a number of disadvantages. A rather large amount of a highly purified R. arrhizus lipase (280 units of lipase to hydrolyze the triglycerides in a 10 microliter serum sample) was required to carry out the complete hydrolysis of the serum triglycerides. The quantities of purified R. arrhizus lipase required, make this procedure prohibitively expensive for routine clinical analysis. In addition, the system disclosed was not suitable for a one-step analysis procedure.
A mixture of a lipase, preferably a microbial lipase, with a protease has also been used to completely hydrolyze serum triglycerides. U.S. Pat. No. 3,703,591. The purity of the lipase used continued to have a significant effect on the efficiency of hydrolysis. U.S. Pat. No. 3,862,009 teaches that the hydrolytic efficiency of R. arrhizus lipase can be improved by carrying out the saponification in the presence of a carboxylesterase and of an alkali metal or alkaline earth metal alkyl sulfate. U.S. Pat. No. 3,898,130 discloses a method for the rapid hydrolysis of triglycerides using a combination of a pancreatic lipase, a microbial lipase, and a bile salt. All three components are essential for the process to operate effectively.